gene therapy II

what is the process of delivering a gene to patient cells?

1. can use viruses or non-viral methods

2. when using viruses, we have to find a way to get the desired gene into the virus

3. use specially engineered eukaryotic cells grown in the lab

• these cells are engineered with genes (trans seqences) that express viral proteins

• packaging cell: supply viral proteins

1. transfer the desired gene linked to the correct packagin sequences (cis sequences) into the packaging cells

2. the packaging cells will start making viruses carrying the desired gene (vector virus)

3. the vector viruses can be collected and used to infect target cells from the patient

• upon infection, the vector virus will deliver the desired gene to the target cell

what are liposomes?

• spherical vesicles composed of synthetic lipid bilayers that mimic the structure of biological membranes
• cationic lipids used for transferring DNA (negatively-charged) into cells
• offer a very versatile option for gene delivery: can accomodate a wide range of DNA encoding both small/large proteins

what is non-viral gene delivery through liposomes?

• the transgene is packaged in vitro with the liposomes and used directly for delivering the DNA to a suitable target tissue in vivo
• the lipid coating protects the DNA in vivo and binds to cells
• the packaged DNA enters cells by direct fusion
• the efficiency of transfer is relatively low, and the introduced foreign gene does not integrate into hosts genome
• high liposome and DNA concentrations required, but both are relatively easy and cheap to manufacture in large amounts

what are the methods of improved targeting of liposomes?

• PEGylated: PEG (improved PK and PD) improves half-life of liposomes
• transferring-conjugated: transferrin can bind transferring receptors found on human cells
• Ab-modified: Ab coating for specificity
• adding a sendal (HVJ) viral protein: allows DNA to escape the endosome into the cytosol intact by disrupting endosomal membrane
• DNA binding proteins (HMG-1): to target foreign DNA into nucleus
• nuclear localizing sequence (NLS) within DNA

what nanoparticles are FDA approved for drug delivery?

• poly-glycolic acid
• poly (lactic-co-glycolic acid): can potentially be used for gene delivery

what are polymer-based nanomaterials for gene delivery?

• electrostatic interactions between DNA and polymer allow nanocomplexes to be formed with DNA, protecting the DNA from degradation
• increasing transfection efficiency
• navigating through the multiple biological barriers

what is naked DNA?

• muscle cells were first shown to be able to take up naked DNA and express proteins
• 99% of the injected DNA is degraded by extracellular nucleases
• large amounts of DNA required
• very low gene transfer efficiency leads to low and transient protein expression
• not immunogenic
• useful when the effect of the expressed protein is naturally amplified
• increase transduction efficiency by electric impulse or "gene gun"

what is a biolistic gene gun?

• uses kinetic energy to deliver nucleic acids inside cells
• DNA is coated on gold or tungsten particles
• 5-10% transuction efficiency
• only be used for exposed targets, such as skin, for local transfection that can withstand pressure
• not useful for deep tissues
• e.g. skeletal myotubules that are hard to transduce with viral vectors due to lack of receptors can be transfected with gene guns

what are the advantages of viral vectors?

• high transfection efficiency
• natural tropism: ability to infect different cell types
• evolved mechanisms for endosomal escape
• natural transportation mechanism of DNA into nucleus

what are the disadvantages of viral vectors?

• strong immune reactions against viral proteins prohibit multiple administrations
• possibility of chromosomal insertion and protooncogene activation
• complication synthesis process
• toxicity, risk of contamination of live virus
• limitation on gene size

what are the advantages of non-viral vectors?

• low immunogenicity
• can be made to be non-toxic
• easy to synthesize: quality control for mass production
• potentially targetable
• no limit on plasmid size
• no integration: can be administered as drugs

what are the disadvantages of non-viral vectors?

• low transfection efficacy
• no natural tropism. endosomal escape, or nuclear transport mechanisms

what is the total number of gene therapy trials currently?

3900

what is the majority of gene types transferred in gene therapy clinical trials?

receptor (29.6%), antigen (16.4%), cytokine (11.5%),…

what is the majority of indications addressed by gene therapy clinical trials?

cancer diseases (68.3%)

what is ADA (adenosine deaminase) deficiency?

• cause of ~20% of cases of severe combined immunodeficiency (SCID)
• patients have multiple severe opportunistic infections, usually beginning in infancy
• inherited as an autosomal RECESSIVE condition
• rare, less than 1/100,000 births
• usually fatal in the first year of life if untreated
• there is selective toxicity to lymphocytes from accumulation of metabolites of deoxyadenosine, especially dATP

what is the rational for considering ADA-SCID for initial gene therapy trials?

• the normal human ADA gene had been cloned
• a single gene defect leads to loss of enzymatic function
• HLA-matched allogeneic bone marrow transplantation completely corrects the disease
• treatment for those patients who lack HLA-matched bone marrow donors is with recombinant ADA protein
• corrected cells have selective growth advantage in vivo
• a small amount of ADA activity is sufficient to correct disease

what was the first human gene therapy trial in 1990?

• 2 girls ages 4 and 9 with ADA SCID were the first gene therapy recepients
• ADA gene delivered into patient lymphocytes using retroviral vectors: ex vivo therapy
• patients continued to receive ADA protein replacement therapy
• immune systems restored

what is X-linked severe combined immunodeficiency (SCID)?

• 40-50% of all SICD cases, absence of functional T and NK cells
• caused by deficiency in γ subunit of cytokine IL-2 receptors (IL2RG gene): also called common γ chain (cγ) as it's a subunit common to the receptors for five other cytokines (IL-4, IL-7, IL-9, IL-15, IL-21)

what happened in gene therapy for X-linked SCID?

• patients received an infusion of autologous bone marrow-derived CD34+ cells transduced with the γ-chain-IL2R
  containing retroviral vector
• gene therapy caused 4 patients to develop leukemia 2-4 years after treatment
  possible reasons:
• something about the vector virus made it integrate at oncogenic sites (LMO2)
• the transferred gene IL2RG itself is oncogenic
• genetic background of patient makes them predisposed to cancer

what were the results of X-linked SCID gene therapy using retroviral vectors?

• improved safety profile: no case of leukemia
• effective immune recovery: functional T cells
• conditioning advancements: incorporated low-dose busulfan to help new stem cells grow; improved recovery of B and NK cells

what is cystic fibrosis?

• major symptoms: impaired lung function, chronic respiratory infections, airway inflammation
• caused by mutations of the CFTR gene affecting chloride ion channel function, leading to dysregulation of epithelial fluid transport in the lung, pancreas, and other organs
• CFTR gene: 250 kbp; consists of 27 exons
• after introns are spliced out the gene, the mRNA is 6100 bp long and is translated into the 1480 AA sequence of CFTR protein
• protein: CF transmembrane conductance regulator
• effects 1 in 3300 caucasians and 1 in 25 are carriers: most common hereditary disease in caucasians
• locus: 7q31.2

what is the CFTR protein?

Cl- channel protein found in membranes of epithelial cells

why is CF an ideal candidate for gene therapy?

• single gene defect
• recessive condition: heterozygous normal, suggesting no gene dosing effects
• main pathology is in the lung: accessible for treatment
• progressive disease with virtually normal phenotype at birth, thereby providing a therapeutic window from symptom in treatment
• protein replacement therapy in lungs not available, and may not be psosible for a membrane protein

what are the requirements for CF gene therapy to work?

• an adequate carrying capacity by the gene delivery system
• to be undetectable by the immune system
• to be non-inflammatory
• to be safe for the patients with pre-existing lung inflammation
• to have an efficiency sufficient to correct the CF phenotype
• to have long duration of expression or the ability to be safely re-administered

what is gene therapy for CF?

• first trials in NY in 1993 using adenoviral vectors were unsuccessful due to significant immune reactions and inflammation in the lungs
• clinical trials are currently ongoing by the UK CF gene therapy consortium: CFTR gene introduced via aerosol to lungs using cationic liposomes

what is hemophilia B?

• christmas disease
• X-linked genetic disorder affecting 1/100,000 male births
• abnormal bleeding due to defective coagulation factor IX: known to be cause of "royal disease"

what is gene therapy for hemophilia B?

• 40% cases are severe
• effective care can be achieved with levels of 75-100 ng/mL factor IX in the blood: symptoms can be significantly reduced by expression of only 1.5-2% of normal levels of factor IX
• factor IX cDNA gene: 1.5 kbp

what are the classifications of factor IX activity?

normal: 100%

• 5 mg/mL
  mild: 5-50%
• >250 ng/mL
• bleed as a result of surgery or major injury
  moderate: 1-5%
• 50-250 ng/mL
• bleed less frequently, once a month
  severe: <1%
• <50 ng/mL
• bleed 1-2 times per week: spontaneous

what is the current treatment for factor IX deficiency?

• factor IX half-life: 18-24 hrsa
• can cost up to $20 million over the lifetime of a patient
• gene therapy, which obviates IV infusions, would advance care both clinically and economically
  infusion of factor IX protein intravenously:
• concentrated from donated human blood
• improved manufacturing and production of rFactor IX has reduced complications of contaminations associated with using a blood product

what is the long-term expression of factor IX after gene therapy using AAV?

• second DNA strand synthesis is rate-limiting step
• self-complementary adeno-associated virus vector (sc-AAV): very efficient, but can only deliver genes that are maximally only ~2.3-2.4 kbp in size
• after infection of target cell, highly efficient gene transfer

what is hemophilia A?

• most common type: 5 times more common than hemophilia B
• X-linked disorder
• factor VIII gene near factor IX on X chromosome: ~9 kbp
• good target for gene therapy as even a low level of secreted protein expression can prevent symptoms

what id hereditary blindness?

• leber's congenital amaurosis (LCA)
• caused by mutation in RPE65 gene
• an eye disorder that primarily affects the retina
• 4 independent clinical trials with AAV2- containing RPE65 reported improvement in vision, with none being associated with vector-related adverse events

what are lysosomal storage diseases?

• more than 50 diseases; most common: gaucher disease, fabry disease, hunter sydrome, hurler syndrome, tay-sachs disease, pompe disease
• the rare, inherited conditions occur when the body doesn't produce the enzymes needed to help break down certain substances, such as fats, sugars, or proteins

what is parkinson's disease?

• cell death in the substantia negra: responsible for making neurotransmitter dopamine
• currently being treated with exogenous dopamine systemically, which inefficiently crosses BBB and has side effects
• gene transduction of tyrosin hydroxylase, enzyme that synthesizes dopamine

what are the strategies for rationality in the gene therapy trials in parkinson's disease?

3 broad approaches to selection of a therapeutic target:

• restoration of dopamine synthesis in the dorsal striatum
• modulation of activity in the basal ganglia downstream of the striatum
• modification fo disease progression by neuroprotection

what are vectors associated with gene therapy for parkinson's disease?

• overexpression of GDNF mediated by the lentiviral vector has conferred some protection of the nigrostriatal dopamine terminals against toxic insults
• Ad-GDNF: can protect dopaminergic neurons and improve dopamine-dependent behavioral function in young rats with progressive 6-OHDA lesions of the nigrostriatal projection
• AAV-2: when administered locally, it transduces only neurons within the CNS and is particularly efficient in brain regions known to be involved in the pathophysiology of parkinson's disease, such as the globus pallidus and substantia nigra

what is restenosis?

• >50% of cases have some extent
• re-narrowing of a coronary artery after treatment with stenting
• common adverse event of endovascular procedures: innovative, less invasive procedure used to treat problems affecting the blood vessel

what is gene-eluting for preventing coronary restenosis/

• stent coated with PLGA-nanoparticles containing VEGF gene in animal model system
• other potential genes to treat restenosis: PCNA (proliferating cell nuclear Ag), NOS

what is gene therapy in cancer?

• engineer T cells to recognize tumor Ag: CEA, NY-ESO-1, CD19
• immunopotentiating genes (IL-2, B7, GM-CSF) introduced into tumor cells to increase immune reaction to tumor
• restoring tumor suppressor genes (e.g. p5, BRCA1, Rb) that are mutated in cancer cells
• anti-sense therapy to turn off oncogenes/replication genes
• suicide genes: implant herpes simplex thymidine kinase into tumor cells

what is CAR T cell therapy?

enhance the natural cancer-fighting ability of patient's own T cells

what is the process of CAR T cell therapy?

1. a sample of a patient's T cells is collected and mixed with viruses carrying several specific genes
2. the viruses deliver these genes to the T cells' nuclei, where they're incorporated into the cells' DNA
3. the genes cause the T cells to express a chimeric Ag receptor (CAR) on their surface
4. CAR directs the T cell to the tumor cell using a specific "address"
5. CAR T cell is then equipped to rapidly destroy the cancer cell
6. when the CAR T cells are infused into the patient, they seek out tumor cells and then proliferate to generate many more cancer-killing cells

what is targeted gene therapy using suicide genes?

• a non-toxic prodrug is administered systemically
• the basic concept: the introduction of viral or bacterial genes into tumor cells, which in turn convert a non-toxic pro-drug to a toxic one
• demostrated limited clinical efficacy for treatment of malignant glioma

what is gene-directed enzyme-producing therapy (GDEPT)?

uses a gene taken from the cancer cell and then modified with other genes to form enzymes that are harmless to healthy cells

what is virus-directed enzyme-prodrug therapy?

uses a virus, such as herpes simplex or cold virus, as the carrier/vector to deliver the modified genes to the cancer cells

what is the cytosine deaminase gene of escherechia coli?

converts the produce 5-fluorocytosine to 5-fluorouracil

what is herpes simplex virus thymidine kinase gene (HSV-tk)?

converts ganciclovir (GCV) to ganciclovir monophosphate, converted by the cancer cells' enzymes to ganciclovir triphosphate

what are the advantages of suicide gene therapy?

• requires short-term gene expression
• safety in clinical trials
• "bystander" tumor killing via gap junction delivery of drug
• enhances sensitivity to conventional therapy

what are the limitations of suicide gene therapy?

• limited spatial distribution of gene transfer vectors
• poor gene transfer efficiency into tumor cells in vivo
• inability to target dispersed tumor cells

what is in vivo CRISPR editing?

• editing genes directly inside the body
• developed treatments that use lipid nanoparticles to deliver CRISPR components to the liver, targeting diseases like transthyretin amyloidosis
• early results show significant reductions in disease-causing proteins

what is base and prime editing?

• allow for precise correction of single-letter genetic mutations without cutting the DNA
• are being explored for treating conditions like progeria and other monogenic diseases, offering potential for safer and more accurate therapies

what are the components of CRISPR-Cas9 system?

RNA-guided DNA endonuclease Cas9 and chimeric single guide RNA (sgRNA)

what is sgRNA?

• has an invariant scaffold region and a space region
• derived from CRISPR RNA (crRNA) and transactivating crRNA
• binds to Cas9 and directs it to the locus of interest by a 20-nt guide sequence via base pairing to the genomic target
• the target sequence in genomic DNA paired to sgRNA sequence is immediately followed by either an NGG/NAG trinucleotide for S. pyogenes-derived Cas9: protospacer adjacent motif (PAM) located on the immediate 3' ends of the sgRNA recongintion sequence

what are the components of the CRISPR-Cas9 complex?

• Cas9 protein: enzyme that acts as molecular scissors; has 2 cuttion domains (HNH and RuvC
• sgRNA: directs Cas9 to a specific location
• PAM: required for CAs9 to bind and cut
• double-strand break: results of Cas9 activity

what is non-homologous end joining (NHEJ)?

• the cell "glues" the broken ends back together
• error-prone and often results in random mutations: small insertions, deletions, substitutions
• useful for gene knockout
• active in both dividing and non-dividing cells

what is homology-directed repair (HDR)?

• if a donor DNA template is provided, the cell can use it as a map to repair the break precisely
• allows for precise gene modification or "knocking in" a specific DNA sequence of interest
• used for gene correction or inserting new genetic material
• primarily active in dividing cells during the G2/S phases of the cell cycle

what are the potential applications and advantages of CRISPR technology?

• altering specific genetic loci through insertions, deletions, point mutations, and sequence inversions
• treating and preventing the spread of disease and improving crops
• transform medicine, enabling us to not only treat but also prevent many diseases
• the system was recently modified to act as a genome regulator, by tethering effector domains to the Cas9 or guide-RNA and as a visualization tool by fusing with marker molecules

what are the potential disadvantages of CRISPR technology?

• philosophical dilemma: ethical concerns about germline gene editing, which are responsible for passing genes on to the next generation
• accuracy: off-target effects, lack of specificity in targeting, and incomplete targeting
• endanger biodiversity: once an organism is modified, they are difficult to distinguish from the wild-type

what is CRISPR-Cas9 engineering of T cells in cancer patients?

• disrupt 3 genes (TRAC, TRBC, PDCD1) to improve antitumor immunity
• a cancer-targeting transgene, NY-ESO-1, was also introduced to recognize tumors

what are cancer vaccines?

• the patient's immune system launches a vigorous attack not only on the newly-infused cancer cells but also on similar cells throughout the body
  this approach involves:
• collecting tumor cells from a patient
• engineering them with genes that cause them to be more conspicuous to the immune system
• re-infusing altered cancer cells into the patient along with an immune-stimulating compound

what are the potential complications of gene therapy?

• gene silencing: repression of promoter
• genotoxicity: complications arising from insertional mutagenesis
• phenotoxicity: complications arising from overexpression or ectopic expression of the transgene
• immunotoxicity: harmful immune response to either the vector/transgene
• risk of horizontal transmission: shedding of infectious vector into the environment
• risk of vertical transmission: germline transmission of donated DNA

what are the strategies to mitigate risks of gene therapy?

• use endogenous cellular promoters, avoid viral-derived regulatory sequences
• use vectors with safer integration profile
• sequence-specific integration
• control transgene expression spatially and temporarily
• carefully monitor T cell reactivity to the vector and transgene to initiate immune suppression if needed
• monitor vector shedding in preclinical models when developing novel vectors
• use of barrier contraceptive methods until vector shedding is negative

what is casgevy (exagamglogene autotemcel)?

• first CRISPR-based gene therapy for treating sickle cell disease
• edits the BCL11A gene in a patient's stem cells to boost fetal Hb production, reducing disease symptoms

what is roctavian (valoctocogene roxaparvovec)?

• one-time gene therapy for severe hemophilia A
• delivers a functional copy of the factor VIII gene using an adeno-associated virus (AAV) vector, enabling patients to produce their own clotting factor

what is hemegenix (etranacogene dezaparvovec)?

• therapy for treating hemophilia B
• uses an AAV vector to deliver the factor IX gene, reducing the need for regular infusions

what is vyjuvek (beremagene geperpavec)?

• first topical gene therapy for treating wounds in patients with dystrophic epidermolysis bullosa
• employs a modified herpes simplex virus to deliver the COL7A1 gene directly to skin cells

what is gendicine?

wild-type p53 gene adenovirus for the treatment of head and neck cancer

what is oncorine?

E1B-defective adenovirus for the treatment of head and neck cancer

what is neovasculogen?

VEGF for the treatment of peripheral arterial disease (PAD) and its complication, critical limb ischemia (CLI)

what is glybera?

adeno-associated virus to deliver a functional copy of the LPL gene to skeletal muscle for the treatment of lipoprotein lipase deficiency (LPLD)

what is imlygic?

genetically modified herpes simplex virus type 1 for the local treament of unresectable lesions in patients with melanoma

what is strimvelis?

stem cell gene therapy to treat ADA-SCID patients

what is kymriah?

• CAR-T therapy for the treatment of acute lymphoblastic leukemia (ALL)
• T cells are genetically modified to hunt down a marker (CD19) that is present on cancerous B cells

what is yescarta?

CAR-T therapy to treat adults with diffuse large B-cell lymphoma: most common form of NHL (non-Hodgkin lymphoma)

what is luxturna?

therapy that uses an AAV vector to deliver a gene (RPE65) into the eye tissue of patients suffering from a rare disease called RPE65 mutation-associated retinal dystrophy

what is zolgensma?

• AAV for spinal muscular atrophy (paralysis, death)
• survival motor neuron 1 mutation